N-(-3-methoxy-5-methylpyrazin-2-yl)-2-(4-′1,3,4-oxadiazol-2-yl-phenyl)pyridine-3 sulphonamide as an anticancer agent

ABSTRACT

The use of N-(3-methoxy-5-methylpyrazin-2-yl)-2-(4-[1,3,4-oxadiazol-2-yl]phenyl)pyridine-3-sulphonamide, or a pharmaceutically acceptable salt thereof, in the treatment of cancer and/or pain in a warm blooded animal such as man is described.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a U.S. National Stage under 35 U.S.C §371 ofInternational Application No. PCT/GB2003/003653 (filed Aug. 20, 2003)which claims priority under 35 U.S.C. §119(a)-(d) to Application No.GB0219660.8 filed on Aug. 23, 2002.

The present application refers toN-(3-methoxy-5-methylpyrazin-2-yl)-2-(4-[1,3,4-oxadiazol-2-yl]phenyl)pyridine-3-sulphonamide,or a pharmaceutically acceptable salt thereof, hereafter “Compound (I)”,and its use in the treatment of cancer in a warm blooded animal such asman. The invention also relates to the use of pharmaceuticalcompositions containing Compound (I), or a pharmaceutically acceptablesalt thereof, in a method of treating cancer in a warm blooded animalsuch as man, and to the use of Compound (I), or a pharmaceuticallyacceptable salt thereof, in the manufacture of medicament for use in amethod of treating cancer in a warm blooded animal such as man. Theinvention also relates to the use of pharmaceutical compositionscontaining Compound (I), or a pharmaceutically acceptable salt thereof,in a method of treating pain in a warm blooded animal such as man, andto the use of Compound (I), or a pharmaceutically acceptable saltthereof, in the manufacture of medicament for use treating pain in awarm blooded animal such as man.

Cancer affects an estimated 10 million people worldwide. This figureincludes incidence, prevalence and mortality. More than 4.4 millioncancer cases are reported from Asia, including 2.5 million cases fromEastern Asia, which has the highest rate of incidence in the world. Bycomparison, Europe has 2.8 million cases, North America 1.4 millioncases, and Africa 627,000 cases.

In the UK and US, for example, more than one in three people willdevelop cancer at some point in their life. Cancer mortality in the U.S.is estimated to account for about 600,000 a year, about one in everyfour deaths, second only to heart disease in percent of all deaths, andsecond to accidents as a cause of death of children 1-14 years of age.The estimated cancer incidence in the U.S. is now about 1,380,000 newcases annually, exclusive of about 900,000 cases of non-melanotic (basaland squamous cell) skin cancer.

Cancer is also a major cause of morbidity in the UK with nearly 260,000new cases (excluding non-melanoma skin cancer) registered in 1997.Cancer is a disease that affects mainly older people, with 65% of casesoccurring in those over 65. Since the average life expectancy in the UKhas almost doubled since the mid nineteenth century, the population atrisk of cancer has grown. Death rates from other causes of death, suchas heart disease, have fallen in recent years while deaths from cancerhave remained relatively stable. The result is that 1 in 3 people willbe diagnosed with cancer during their lifetime and 1 in 4 people willdie from cancer. In people under the age of 75, deaths from canceroutnumber deaths from diseases of the circulatory system, includingischaemic heart disease and stroke. In 2000, there were 151,200 deathsfrom cancer. Over one fifth (22 per cent) of these were from lungcancer, and a quarter (26 per cent) from cancers of the large bowel,breast and prostate.

Worldwide, the incidence and mortality rates of certain types of cancer(of stomach, breast, prostate, skin, and so on) have wide geographicaldifferences which are attributed to racial, cultural, and especiallyenvironmental influences. There are over 200 different types of cancerbut the four major types, lung, breast, prostate and colorectal, accountfor over half of all cases diagnosed in the UK and US. Prostate canceris the fourth most common malignancy among men worldwide, with anestimated 400,000 new cases diagnosed annually, accounting 10 for 3.9percent of all new cancer cases.

Current options for treating cancers include surgical resection,external beam radiation therapy and/or systemic chemotherapy. These arepartially successful in some forms of cancer, but are not successful inothers. There is a clear need for new therapeutic treatments.

Non-steroidal anti-inflammatory drugs SAIDS) and opiates are the mainclasses of drugs in pain relief. However both possess undesirable sideeffects. NSAIDS are known to cause gastrointestinal irritation andopiates are known to be addictive. There is thus also a clear need fornew treatments for the management and treatment of pain.

Recently, endothelin A receptor antagonists have been identified aspotentially of value in the treatment of cancer (Cancer Research, 56,663-668, Feb. 15^(th), 1996 and Nature Medicine, Volume 1, Number 9,September 1999, 944-949).

The endothelins are a family of endogenous 21 amino acid peptidescomprising three isoforms, endothelin-1 (ET-1), endothelin-2 andendothelin-3. The endothelins are formed by cleavage of the Trp²¹-Val²²bond of their corresponding proendothelins by an endothelin convertingenzyme. The endothelins are among the most potent vasoconstrictors knownand have a characteristic long duration of action. They exhibit a widerange of other activities including cell proliferation and mitogenesis,extravasation and chemotaxis, and also interact with a number of othervasoactive agents.

The endothelins are released from a range of tissue and cell sourcesincluding vascular endothelium, vascular smooth muscle, kidney, liver,uterus, airways, intestine and leukocytes. Release can be stimulated byhypoxia, shear stress, physical injury and a wide range of hormones andcytokines. Elevated endothelin levels have been found in a number ofdisease states in man including cancers.

The present invention concerns the surprising finding that Compound (I)is a particularly potent anti-cancer agent. Compound (I) is described asan endothelin receptor antagonist in WO96/4068 1, and although inWO96/40681 it is acknowledged that elevated endothelin levels have beenfound in a number of disease states in man including certain cancers,there is no hint or suggestion that this compound would possess theparticular beneficial efficacious, metabolic and toxicological profilesthat makes it such a potent anti-cancer agent. WO96/4068 1 claims theendothelin receptors described therein solely for cardiovasculardiseases. For example in the introduction it is stated these compoundsare useful in the treatment of diseases or medical conditions including“hypertension, pulmonary hypertension, cardiac or cerebral circulatorydisease and renal disease”. The claims list the following medicaldisease states “hypertension, pulmonary hypertension, congestive heartfailure, dyslipidaemia, atherosclerosis, restenosis, acute and chronicrenal failure, ischaemic stroke, subarachnoid haemorrhage, intermittentclaudication, critical limb ischaemia, asthma or organ failure aftergeneral surgery or transplantation”. There is no hint or suggestion fromWO96/40681 that this compound would possess the particular beneficialefficacious, metabolic and toxicological profiles that makes it such apotent anti-cancer agent. In fact, the present inventors havesurprisingly established that Compound (I) is a specific endothelin-A(ET_(A)) antagonist and has no measurable activity against endothelin-B(ET_(B)).

The ET_(A) receptor has been shown, via a variety of mechanisms, to bethe more important pathological receptor of the two identifiedendothelin receptors in oncology: in the reduction of abnormal cellproliferation (Bagnato et. al., (1995), Clin Cancer Res 1, 1059-1066);as a anti-apoptotic (Wu Wang et. al., (1997), Biochem J,. 328, 733-737);as an anti-angiogenic agent (Spinella et al., (2002), J. Biol. Chem,227(31), 27850-27855); and as an inhibitor of bone metastases (Guise et.al., ASCO (2000) abstract 331 and Nelson, et. al., (1999), Urology 53,1063-1069) in addition to mediating pain which is a common co-morbidityin cancer. It has been shown (Dahlof et al., (1990), J Hypertens, 8,811-817) that large doses of endothelin-1 causes pain, and causes painsensitization, but that this can be inhibited by an ETA antagonist (e.g.Davar et al., (1998), Neuroreport 9, 2279-2283 and De Mello et al.,(1998), Pain, 77, 261-269). Therefore in another aspect of theinvention, Compound (I) is administered for the prevention or treatmentof pain mediated by the endothelin system, in particular that associatedwith elevated endothelin-1 levels.

Conversely, there is emerging evidence (e.g. Cattaruzza et. al., (2002),FASEB J. 14(7), 991-998 and Okazawa et. al., (1998), J Biol Chem, 273,12581-12592) that the ET_(B) receptor is involved in apoptoticsignalling. The blocking of pro-apoptotic pathways would be undesirablein the treatment of cancer, hence a compound that specifically targetedthe ET_(A) receptor while leaving the ET_(B) receptor unaffected wouldbe of the greatest utility in the treatment of cancer. Compound (I) issuch a compound.

Compound (I) by acting specifically on the ET_(A) receptor has manyadvantages over endothelin antagonists that also have measurable ET_(B)activity. For instance Compound (I) could be administered to a patientwithout the administrator or prescribing medical practitioner needing totitrate the dose of Compound (I) looking for signs of ET_(B) activity(for example oedema). Furthermore, larger doses could potentially beadministered because there would be no ET_(B) side effects.

Another disadvantage of ET_(B) inhibition is that it causes a rise inplasma endothelin. Potentially, over the course of treatment, for amixed ET_(A)/ET_(B) inhibitor, or a compound that selectively targetedthe ET_(A) receptor, but still had measurable ET_(B) activity, thiswould result in increasingly larger doses of inhibitor being needed tohave the same beneficial ET_(A) effects. A specific ET_(A) inhibitorwould not encounter this problem.

Therefore according to the present invention, there is provided Compound(I), or a pharmaceutically acceptable salt thereof, for use in thetreatment of cancer in a warm blooded animal such as man.

In one aspect, where Compound (I), or a pharmaceutically acceptable saltthereof, is referred to this refers to the compound only. In anotheraspect this refers to a pharmaceutically acceptable salt of Compound(I).

According to another feature of the present invention, there is providedCompound (I), or a pharmaceutically acceptable salt thereof, in themanufacture of a medicament for use in the treatment of cancer in a warmblooded animal such as man.

According to a further feature of this aspect of the invention there isprovided a method of treating cancer which comprises administering aneffective amount of Compound (I), or a pharmaceutically acceptable saltthereof, to a warm blooded animal such as man.

According to a further feature of this aspect of the invention there isprovided a pharmaceutical composition which comprises Compound (I), or apharmaceutically acceptable salt thereof, in association with apharmaceutically acceptable diluent or carrier for use in the treatmentof cancer in a warm blooded animal such as man.

In another aspect of the invention there is provided the use of Compound(I), or a pharmaceutically acceptable salt thereof, in the reduction ofabnormal proliferation in a cancerous cell or inducing differentiationof a cancerous cell in a warm blooded animal such as man.

In another aspect of the invention there is provided the use of Compound(I), or a pharmaceutically acceptable salt thereof, in the manufactureof a medicament for use in the reduction of abnormal proliferation in acancerous cell or inducing differentiation of a cancerous cell in a warmblooded animal such as man.

In another aspect of the invention there is provided a method forreducing abnormal proliferation in a cancerous cell or inducingdifferentiation of a cancerous cell which comprises administering aneffective amount of Compound (I), or a pharmaceutically acceptable saltthereof, to a warm blooded animal such as man.

According to a further feature of this aspect of the invention there isprovided a pharmaceutical composition which comprises Compound (I), or apharmaceutically acceptable salt thereof, in association with apharmaceutically acceptable diluent or carrier for use in the reductionof abnormal proliferation in a cancerous cell or inducingdifferentiation of a cancerous cell in a warm blooded animal such asman.

In another aspect of the invention there is provided the use of Compound(I), or a pharmaceutically acceptable salt thereof, in inducingapoptosis in a cancerous cell in a warm blooded animal such as man.

In another aspect of the invention there is provided the use of Compound(I), or a pharmaceutically acceptable salt thereof, in the manufactureof a medicament for use in inducing apoptosis in a cancerous cell in awarm blooded animal such as man.

In another aspect of the invention there is provided a method ofinducing apoptosis in a cancerous cell which comprises administering aneffective amount of Compound (I), or a pharmaceutically acceptable saltthereof, to a warm blooded animal such as man.

According to a further feature of this aspect of the invention there isprovided a pharmaceutical composition which comprises Compound (I), or apharmaceutically acceptable salt thereof, in association with apharmaceutically acceptable diluent or carrier for use in inducingapoptosis in a cancerous cell in a warm blooded animal such as man.

In another aspect of the invention there is provided the use of Compound(I), or a pharmaceutically acceptable salt thereof, as ananti-angiogenic and vascular targeting agent in blood vessels supplyinga cancerous cell in a warm blooded animal such as man.

In another aspect of the invention there is provided the use of Compound(I), or a pharmaceutically acceptable salt thereof, in the manufactureof a medicament for use as an anti-angiogenic and vascular targetingagent in blood vessels supplying a cancerous cell in a warm bloodedanimal such as man.

In another aspect of the invention there is provided a method ofproviding an anti-angiogenic and vascular targeting agent in bloodvessels supplying a cancerous cell which comprises administering aneffective amount of Compound (I), or a pharmaceutically acceptable saltthereof, to a warm blooded animal such as man.

According to a further feature of this aspect of the invention there isprovided a pharmaceutical composition which comprises Compound (I), or apharmaceutically acceptable salt thereof, in association with apharmaceutically acceptable diluent or carrier for use as ananti-angiogenic and vascular targeting agent in blood vessels supplyinga cancerous cell in a warm blooded animal such as man.

By the term “vascular targeting agent” it is to be understood that thesite of action of Compound (I) would be on the vasculature itself ratherthan the tumour.

In another aspect of the invention there is provided the use of Compound(I), or a pharmaceutically acceptable salt thereof, as ananti-angiogenic agent in a warm blooded animal such as man.

In another aspect of the invention there is provided the use of Compound(I), or a pharmaceutically acceptable salt thereof, in the manufactureof a medicament for use as an anti-angiogenic agent in a warm bloodedanimal such as man.

In another aspect of the invention there is provided a method ofproviding an anti-angiogenic effect which comprises administering aneffective amount of Compound (I), or a pharmaceutically acceptable saltthereof, to a warm blooded animal such as man.

According to a further feature of this aspect of the invention there isprovided a pharmaceutical composition which comprises Compound (I), or apharmaceutically acceptable salt thereof, in association with apharmaceutically acceptable diluent or carrier for use as ananti-angiogenic agent in a warm blooded animal such as man.

In another aspect of the invention there is provided the use of Compound(I), or a pharmaceutically acceptable salt thereof, as an inhibitor ofbone metastases and an inhibitor of invasion in a warm blooded animalsuch as man.

In another aspect of the invention there is provided the use of Compound(I), or a pharmaceutically acceptable salt thereof, in the manufactureof a medicament for use as an inhibitor of bone metastases and aninhibitor of invasion in a warm blooded animal such as man.

In another aspect of the invention there is provided a method ofinhibiting bone metastases and inhibiting invasion which comprisesadministering an effective amount of Compound (I), or a pharmaceuticallyacceptable salt thereof, to a warm blooded animal such as man.

According to a further feature of this aspect of the invention there isprovided a pharmaceutical composition which comprises Compound (I), or apharmaceutically acceptable salt thereof, in association with apharmaceutically acceptable diluent or carrier for use as an inhibitorof bone metastases and an inhibitor of invasion in a warm blooded animalsuch as man.

In another aspect of the invention there is provided the use of Compound(I), or a pharmaceutically acceptable salt thereof, as an inhibitor ofbone metastases in a warm blooded animal such as man.

In another aspect of the invention there is provided the use of Compound(I), or a pharmaceutically acceptable salt thereof, in the manufactureof a medicament for use as an inhibitor of bone metastases in a warmblooded animal such as man.

In another aspect of the invention there is provided a method ofinhibiting bone metastases which comprises administering an effectiveamount of Compound (I), or a pharmaceutically acceptable salt thereof,to a warm blooded animal such as man.

According to a further feature of this aspect of the invention there isprovided a pharmaceutical composition which comprises Compound (I), or apharmaceutically acceptable salt thereof, in association with apharmaceutically acceptable diluent or carrier for use as an inhibitorof bone metastases in a warm blooded animal such as man.

In another aspect of the invention there is provided the use of Compound(I), or a pharmaceutically acceptable salt thereof, in the prevention ofbone metastases in a warm blooded animal such as man.

In another aspect of the invention there is provided the use of Compound(I), or a pharmaceutically acceptable salt thereof, in the manufactureof a medicament for use in the prevention of bone metastases in a warmblooded animal such as man.

In another aspect of the invention there is provided a method ofpreventing bone metastases which comprises administering an effectiveamount of Compound (I), or a pharmaceutically acceptable salt thereof,to a warm blooded animal such as man.

According to a further feature of this aspect of the invention there isprovided a pharmaceutical composition which comprises Compound (I), or apharmaceutically acceptable salt thereof, in association with apharmaceutically acceptable diluent or carrier for use in the preventionof bone metastases in a warm blooded animal such as man.

In another aspect of the invention there is provided the use of Compound(I), or a pharmaceutically acceptable salt thereof, in the treatment ofbone metastases in a warm blooded animal such as man.

In another aspect of the invention there is provided the use of Compound(I), or a pharmaceutically acceptable salt thereof, in the manufactureof a medicament for use in the treatment of bone metastases in a warmblooded animal such as man.

In another aspect of the invention there is provided a method oftreating bone metastases which comprises administering an effectiveamount of Compound (I), or a pharmaceutically acceptable salt thereof,to a warm blooded animal such as man.

According to a further feature of this aspect of the invention there isprovided a pharmaceutical composition which comprises Compound (I), or apharmaceutically acceptable salt thereof, in association with apharmaceutically acceptable diluent or carrier for use in the treatmentof bone metastases in a warm blooded animal such as man.

In a further aspect of the invention, there is provided the inhibition,treatment and/or prevention of bone metastases, as described herein,wherein the bone metastases are as a result of renal, thyroid, lung,breast or prostate cancer.

In another aspect of the invention there is provided the use of Compound(I), or a pharmaceutically acceptable salt thereof, in the prevention ortreatment of pain associated with elevated endothelin-1 production in awarm blooded animal such as man.

In another aspect of the invention there is provided the use of Compound(I), or a pharmaceutically acceptable salt thereof, in the manufactureof a medicament for use in the prevention or treatment of painassociated with elevated endothelin-1 production in a warm bloodedanimal such as man.

In another aspect of the invention there is provided a method oftreating pain associated with elevated endothelin-1 production whichcomprises administering an effective amount of Compound (I), or apharmaceutically acceptable salt thereof, to a warm blooded animal suchas man.

According to a further feature of this aspect of the invention there isprovided a pharmaceutical composition which comprises Compound (I), or apharmaceutically acceptable salt thereof, in association with apharmaceutically acceptable diluent or carrier for use in the preventionor treatment of pain associated with elevated endothelin-1 production ina warm blooded animal such as man.

In another aspect of the invention there is provided the use of Compound(I), or a pharmaceutically acceptable salt thereof, in the prevention ortreatment of pain in a warm blooded animal such as man.

In another aspect of the invention there is provided the use of Compound(I), or a pharmaceutically acceptable salt thereof, in the manufactureof a medicament for use in the prevention or treatment of pain in a warmblooded animal such as man.

In another aspect of the invention there is provided a method oftreating pain which comprises administering an effective amount ofCompound (I), or a pharmaceutically acceptable salt thereof, to a warmblooded animal such as man.

In another aspect of the invention there is provided the use of Compound(I), or a pharmaceutically acceptable salt thereof, in the prevention ortreatment of pain associated with stimulation of the ET_(A) receptor ina warm blooded animal such as man.

In another aspect of the invention there is provided the use of Compound(I), or a pharmaceutically acceptable salt thereof, in the manufactureof a medicament for use in the prevention or treatment of painassociated with stimulation of the ET_(A) receptor in a warm bloodedanimal such as man.

In another aspect of the invention there is provided a method oftreating pain associated with stimulation of the ET_(A) receptor whichcomprises administering an effective amount of Compound (I), or apharmaceutically acceptable salt thereof, to a warm blooded animal suchas man.

Where cancer is referred to, particularly it refers to oesophagealcancer, myeloma, hepatocellular, pancreatic, cervical cancer, ewingstumour, neuroblastoma, Kaposis sarcoma, ovarian cancer, breast cancer,colorectal cancer, prostate cancer, bladder cancer, melanoma, lungcancer—non small cell lung cancer (NSCLC), and small cell lung cancer(SCLC)—gastric cancer, head and neck cancer, renal cancer, lymphoma andleukaemia. More particularly it refers to prostate cancer. In addition,more particularly it refers to SCLC, NSCLC, colorectal cancer, ovariancancer and/or breast cancer. In addition, more particularly it refers toSCLC. In addition, more particularly it refers to NSCLC. In addition,more particularly it refers to colorectal cancer. In addition, moreparticularly it refers to ovarian cancer. In addition, more particularlyit refers to breast cancer. Furthermore, more particularly it refers tobladder cancer, oesophageal cancer, gastric cancer, melanoma, cervicalcancer and/or renal cancer. In addition it refers to endometrial, liver,stomach, thyroid, rectal and/or brain cancer. In another aspect of theinvention, the cancer is not melanoma. In another embodiment of theinvention, particularly the cancer is in a metastatic state, and moreparticularly the cancer produces metastases to the bone. In a furtherembodiment of the invention, particularly the cancer is in a metastaticstate, and more particularly the cancer produces skin metastases. In afurther embodiment of the invention, particularly the cancer is in ametastatic state, and more particularly the cancer produces lymphaticmetastases. In a further embodiment of the invention, the cancer is in anon-metastatic state.

It is to be understood that when the cancer is in a metastatic state,that Compound (I) acts at both the primary tumour site and themetastases. Compound (I) both prevents, treats and inhibits metastases.

In one aspect of the invention, where pain is referred to, this is painassociated with raised endothelin-1 levels. In another aspect of theinvention this is pain associated with stimulation of the ET_(A)receptor resulting from situations where ET_(B) down-regulation hasoccurred leading to abnormal ET_(A) stimulation and/or elevation ofendothelin-1 levels. Particularly this is pain associated with cancer.More particularly it is pain associated with prostate cancer.

According to a further feature of this aspect of the invention there isprovided a pharmaceutical composition which comprises Compound (I), or apharmaceutically acceptable salt thereof, in association with apharmaceutically acceptable diluent or carrier for use in the preventionor treatment of pain associated with stimulation of the ET_(A) receptorin a warm blooded animal such as man.

Additionally, Compound (I) is expected to be useful in the treatmentand/or prophylaxis of pain of different origins and causes, includingacute as well as chronic pain states. Examples are pain caused bychemical, mechanical, radiation (including sunburn), thermal (includingburns), infectious or inflammatory tissue trauma or cancer,postoperative pain, postpartum pain, the pain associated with jointconditions (such as rheumatoid arthritis and osteoarthritis), painassociated with dental conditions (such as dental caries andgingivitis), myofascial and low back pain, pain associated with bonedisorders (such as osteoporosis, hypercalcaemia of malignancy andPaget's disease) and the pain associated with sports injuries andsprains.

Also neuropathic pain conditions of central or peripheral origin couldbe treated or prevented with Compound (I). Examples of these painconditions are pain associated with trigeminal neuralgia, painassociated with postherpetic neuralgia (PHN), pain associated withdiabetic mono/poly neuropathy, pain associated with nerve trauma, painassociated with spinal cord injury, pain associated with central poststroke, pain associated with multiple sclerosis and pain associated withParkinson's disease.

Other pain states of visceral origin such as caused by ulcer,dysmenorrhea, endometriosis, irritable bowel syndrome, dyspepsia, pelvicpain etc. could also be treated or prevented with Compound (I).

Additionally, Compound (I) is expected to be useful in the treatmentand/or prophylaxis of additional types of pain for example complexregional pain syndrome, vasospastic/ischemic pains (e.g. Raynaudsyndrome) and bone pain.

A further aspect of the invention is to use Compound (I) for oraltreatment of neuropathic or central pain states.

Suitable pharmaceutically-acceptable salts include, for example, saltswith alkali metal (such as sodium, potassium or lithium), alkaline earthmetals (such as calcium or magnesium), ammonium salts, and salts withorganic bases affording physiologically acceptable cations, such assalts with methylamine, dimethylamine, trimethylamine, piperidine andmorpholine. In addition, suitable pharmaceutically-acceptable saltsinclude, pharmaceutically-acceptable acid-addition salts with hydrogenhalides, sulphuric acid, phosphoric acid and with organic acids such ascitric acid, maleic acid, methanesulphonic acid and p-toluenesulphonicacid.

LEGENDS TO FIGURES

FIG. 1: This is a Western Blot showing inhibition of ET-1 induced MAPKphosphorylation with Compound (I) in the osteoblast cell lineMC3T3.E1/J1 from study 2 below. The proteins have been run on a gel thentransferred over to a nitrocellulose membrane, where they are probed forusing the primary and secondary antibodies. The following abbreviationsare used:

-   SCM: serum containing media-   SFM: serum free media

FIG. 2: This is a graph depicting, inhibition of ET-1 induced MAPKphosphorylation with Compound (I) in the osteoblast cell line MC3T3.E1/J1 also from study 2.

The following in vivo and in vitro studies can be used to determine theefficacy of Compound (I) in oncology.

1) Endothelin Human Receptor Binding Assay

Human recombinant ET_(A) or ET_(B) receptors were expressed in mouseerythroleukaemic (MEL) cells and membranes prepared for competitionbinding studies using ¹²⁵I-labelled ET-1 as the radioligand. Incubationswere carried out in triplicate in the presence of Compound (I),10⁻¹⁰-10⁻⁴ M in half log increments, and inhibition of ET-1 binding wasexpressed as a geometric mean pIC₅₀ value with 95% confidence limits.

Results

The pIC₅₀ (negative log of the concentration of compound required todisplace 50% of the ligand) for Compound (I) at the ET_(A) receptor was8.27 [8.23-8.32] (n=4). Displacement curves were normal with slopesclose to unity. Compound (I) had no measurable affinity for the ET_(B)receptor with a mean displacement of 1.2±0.7% (n=3) at a concentrationof 10⁻⁴M, a figure well within the limits of sensitivity of the assay.

Conclusion

Compound (I) is a high affinity ligand for the human ET_(A) receptor andis ET_(A) specific, having no significant ET_(B) receptor affinity.

2) Compound (I) as a Treatment for Metastatic Cancer: OsteoblastData—Inhibition of ET-1 Induced MAPK Stimulation with Compound (I)

Compound (I) may well have a role in the treatment of not only primarytumours but also metastatic tumours and the pathological production ofnew bone in and around metastatic deposits. Described below is anexperiment demonstrating the utility of Compound (I) in treating theosteoblastic bone pathology.

The important clinical pathology seen in the bone metastatic regions ofpatients with advanced prostate cancer presents as an inappropriateosteoblastic stimulation, i.e. the presence of prostatic tumourmetastases in bone results in the net production of new bone andeventually an increase in bone density around the metastatic deposit(reviewed in Cancer Metastasis Rev. 2001; 20(34):33349). Thehypothesised mechanism behind this pathology is a release of ET-1 fromthe metastatic prostate cell in the early establishment of the secondarybone tumour.

ET-1 stimulation of the osteoblast has been described as the key step inthe pathological formation of new bone in prostate bone metastasis(Invest New Drugs. 2002; 20(2):173-82). It has been shown that ET-1 actsto directly induce proliferation and differentiation of the osteoblast,as well as stimulate the osteoblast to produce other growth factors, bysimulation of the ET_(A) receptor and subsequent phosphorylation of MAPkinase (Bone. 1999; 24(4):315-20 and J Bone Miner Res. 2002;17(10):1774-84). In this way stimulation of the ET_(A) receptor causesboth growth of bone and also, by release of growth factors into thelocal environment, survival and growth of the metastatic tumour cell.The tumour cells and osteoblastic cells in a metastatic deposittherefore participate in a “vicious cycle” in which their proliferativeresponses support each other, overcoming the normal regulatorymechanisms which control and limit bone formation (Nat Rev Cancer. 2002;2(8): 584-93).

In the experiments described below the present inventors firstdemonstrate the ability of ET-1 to stimulate MAP kinase in osteoblasticcells. This stimulation promotes proliferation of the cells andactivation of the pathways shown to be important in the release ofgrowth factors from the osteoblast.

The inventors then demonstrate that Compound (I), an ET_(A) antagonistis an effective antagonist of this ET-1 stimulation.

Method

The MC3T3. E1/J1 cell line was isolated from a parental cell line,MC3T3-E1 (available from Invitrogen), which had in turn been derivedfrom newborn C57BL/6 mouse calvaria. The MC3T3 E1/J1 line is describedas an osteoblastic line. To initiate the experiments described below,MC3T3.E1/J1 cells were plated at a density of 2.4×10⁴ cells/well (24well plates) in serum containing media and incubated for 48 hours. Thecells were washed twice in PBS and re-incubated for approximately 17hours in serum starvation media.

At this stage, cells were then incubated with or without Compound (I)for 30 minutes then stimulated with growth factor (PDGF or ET-1) for 3minutes. All media was then removed and the cells lysed and stored at−20° C. for electrophoresis/western blot, which localized phosphorylatedMAPK and phosphorylated Akt probing with anti-phospho-p44/42 MAPK (Thr202/204) and anti-Phospho AKT (Ser 473) antibodies (both commerciallyavailable from Cell Signalling Technology). The protein bands werequantitated by densitometry, and plotted as arbitrary densitometryunits. Phosphorylated MAPK levels were normalised to total MAPK levels.

Results

Stimulation of cells with ET-1 for 3 minutes resulted in increasedphosphorylation of MAPK in the osteoblast cell line MC3T3.E1/J1.Stimulation of the cells with a standard growth factor, PDGF, alsoresulted in increased phosphorylation of MAPK. Compound (I) inhibitedET-1-induced MAPK phosphorylation in osteoblasts.

TABLE 1 Inhibition of ET-1 induced MAPK phosphorylation with Compound(I) in the osteoblast cell line MC3T3.E1/J1 Environment Average CompleteMedia 151.70 Serum Free Media 100.00 ET-1 100 nm 312.78 ET-1 100 nm369.85 +Compound (I) 20 μm 109.18 +Compound (I) 10 μm 105.15 +Compound(I) 1 μm 157.41 +Compound (I) 0.1 μm 422.11This data is represented in FIGS. 1 and 2.

Note: The above experiment is not reliant on use of the particularMC3T3.E1/J1 cell line, it could, for example, be performed using thecommercially available parental cell line MC3T3-E1.

3) Compound (I) as an Inhibitor of Angiogenesis

ET_(A) receptor activation by ET-1 contributes to tumour growth andprogression, mediated by various mechanisms in the literature to suggestthat specifically inhibiting ET_(A) will produce beneficial effects onprimary tumours quite separate to its effects on bone metastases. Thesemechanisms include anti-apoptosis, direct and indirect growth promotionand promotion of cell motility (Nat Rev Cancer. 2003; 3(2): 110-6).

Of more recent and increasing interest is the role of ET-1 mediated bythe ET_(A) receptor as key players in tumour angiogenesis (J CardiovascPharmacol. 2000; 36: S135-9). Mechanistic studies have now shown thatthe ET_(A) receptor is important in the production of the potentangiogenic factor VEGF (Life Sci. 1998; 63(6): 477-84) by directinduction of a hypoxia-inducible factor, HIF-1α (J Biol Chem. 2002; 277:27850-5). The increasing literature to support the role of endothelinand the ET_(A) receptor in tumour angiogenesis was reviewed veryrecently by Bagnato and Spinella, (Trends Endocrinol Metab. 2003; 14(1):44-50).

In the experiment described below we show the effect of Compound (I) onthe angiogenesis induced by newly formed tumours following human tumourcell inoculation in animal models.

Method

Tumour cells were inoculated intra-dermally in nude mice, Compound (I)25 or 50 mg/kg or vehicle was given once daily p.o. with the first dosegiven on the day after cell implantation and the mice were sacrificed 5days later. A 1 cm² area with the tumour at the centre was examined andthe number of blood vessels bifurcations within that area supplying thetumour were counted. The number of vessels supplying tumours fromanimals treated with test drug and vehicle were compared and the effectof Compound (I) was calculated as a percentage reduction of vesselcount.

Results

Compound (I) caused reductions in blood vessel density around tumours intreated animals compared to vehicle controls. Reductions in vesselcounts by Compound (I) were seen around tumours induced by both colonand prostate cell lines in five in vivo studies.

TABLE 2 Inhibition of angiogenesis in primary tumours caused by Compound(I) Compound (I) Inhibition of vessel Cell Line Tumour Type dose (mg/kg)count ¹ LOVO Colon 50 20% (P = 0.001) LOVO Colon 50 28% (P < 0.001) LOVOColon 25 28% (P < 0.001) DU145 Prostate 50 30% (P < 0.05) DU145 Prostate25 38% (P < 0.001) ¹ statistically analyzed by the ANOVA test comparedto vehicle controls The above cells lines are commercially available.One source is the ATCC (American Type Culture Collection). LOVO has ATCCNo = CCL-229. DU145 has ATCC No = HTB-81.Discussion

We have shown that, in vitro, Compound (I) is an effective inhibitor ofET-1 mediated activation of MAP kinase in osteoblasts as well as beingeffective in inhibiting angiogenesis in primary tumours in vivo. Thisconfirms the potential for this agent as a therapy in metastaticprostate cancer as it may have beneficial effects in preventingpathological bone density increases (by inhibition of osteoblasticproliferation), mediated by MAPK pathway as well as inhibiting therelease of growth factors which support the survival and growth oftumour cells in the bone microenvironment in addition toanti-angiogenesis effect at the primary tumour.

4) Compound (I) as an Endothelin Receptor Antagonist in the HumanEndothelin System

Human forearm blood flow can be assessed by temporarily impeding thevenous drainage from the arm by the application of a pneumatic cuff onthe upper arm, which is then inflated to just above venous pressure. Theresulting arterial flow into the arm with no corresponding venousdrainage leads to engorgement and swelling of the forearm, which can bedetected with sensitive strain gauges. Infusion of the arterialvasoconstrictor ET-1 into the brachial artery leads to a reduction inforearm distension due to decreased arterial inflow. Thisvasoconstriction is mediated via endothelin receptors on the vascularendothelium and associated smooth muscle.

Method

A study was performed to investigate the ability of Compound (I) toantagonise the vasoconstrictor effect of ET-1 via endothelin receptorsin this model in healthy male subjects aged 18-65. Eight subjectsreceived single oral doses of 10 mg Compound (I), 30 mg Compound (I) andplacebo in a randomised, double blind manner on study days at least 7days apart. Forearm vasoconstriction in response to ET1 was assessedbetween 2 and 4 hours post dosing with Compound (I).

Results

Overall, Compound (I) produced a statistically significant reduction inforearm blood flow in response to infused ET-1 compared to placebo(p=0.0210) with evidence of a dose response between the dosesinvestigated. This demonstrates that Compound (I) is an endothelinreceptor antagonist in the human endothelin system.

5) Compound (I) in a Dose-Escalation Study to Assess the Tolerabilityand Pharmacokinetics of Compound (I) given Orally Once Daily in PatientWith Metastatic Prostate Cancer

The following study can be undertaken to determine the maximum welltolerated dose (MWTD) of Compound (I) in subjects with metastaticprostate cancer. This study will allow you to observe the effect ofCompound (I) on prostate-specific antigen (PSA), observe the effect ofCompound (I) on a serological biomarker of bone metastasis and provideyou with pharmacokinetic characterization of Compound (I) in subjectswith metastatic prostate cancer.

Method

Patients with prostate cancer who have documented bone metastases(confirmed by bone scan within 3 months of study entry) can be used forthis study. Compound (I) can be given orally once daily in tablet form.120 mg can be used as the starting dose. Subjects can be given studymedication for 28 days or until withdrawal criteria are met. Each doselevel can recruit up to three subjects with metastatic prostate cancer.

A formal assessment of tolerability can be made in each subjectfollowing one week of Compound (I) administration. Dose escalation canoccur when two subjects in any cohort have not experienced a doselimiting toxicity (DLT) following one week of continuous Compound (I)administration. The dose can escalate by a factor of two at each step.If one subject at a particular dose level has a DLT, then two additionalsubjects at the same dose level must not experience DLTs in order toescalate to the next dose level.

Subjects can continue therapy for twenty eight days unless thewithdrawal criteria are met. When a minimum of two subjects in anycohort have been given a dose that is considered to be not welltolerated at any time point after administration, dose escalation willend, and the closest dose below this will be taken as the MWTD.

The following outcomes can be observed:

Incidence and severity of adverse events;

PSA concentration (total and ratio of free to total) at 1, 2, and 4weeks in subjects treated with Compound (I);

Change in PSA (total and ratio of free to total) from before Compound(I) administration to 1, 2 and 4 weeks after Compound (I)administration;

Change in a serum marker of bony metastatic involvement (bone alkalinephosphatase) from the level before Compound (I) administration to levelsafter 1, 2, and 4 weeks of Compound (I) administration; and

Plasma concentrations and variables of Compound (I) following a singledose and multiple doses at steady state.

Testing for Pain Relief

The analgesic effect of Compound (I) may be measured, for example, inthe murine model of cancer pain described by Wacnik et al., Journal ofNeuroscience (2001), 21, 9355.

In a further embodiment of the present invention Compound (I), or apharmaceutically acceptable salt thereof, is administered to a cell orindividual prior to the development of cancer. For example, a person atrisk of developing cancer may be treated with Compound (I), or apharmaceutically acceptable salt thereof, to prevent or inhibit thedevelopment of cancer and/or to prevent the development of metastases.

Compound (I), or a pharmaceutically acceptable salt thereof, can beadministered for therapeutic or prophylactic use to a warm bloodedanimal such as man by methods known in the art. Administration can occurdirectly at the tumour site, or particularly, systemic administration.

Compound (I), or a pharmaceutically acceptable salt thereof, can beadministered for therapeutic or prophylactic use to a warm bloodedanimal such as man in the form of conventional pharmaceuticalcompositions. The composition may be in a form suitable for oraladministration, for example as a tablet or capsule, for parenteralinjection (including intravenous, subcutaneous, intramuscular,intravascular or infusion) as a sterile solution, suspension oremulsion, for topical administration as an ointment or cream or forrectal administration as a suppository. In general the abovecompositions may be prepared in a conventional manner using conventionalexcipients. For example, Compound (I) can be formulated as a tabletusing the following excipients:

-   -   Compound (I);    -   Lactose monohydrate (filler);    -   Croscarmellose sodium (disintegrant);    -   Povidone (binder);    -   Magnesium stearate (lubricant);    -   Hypromellose (film coat component);    -   Polyethylene glycol 300 (film coat component); and    -   Titanium dioxide (film coat component).

The amount of Compound (I), or a pharmaceutically acceptable saltthereof, administered would be that sufficient to provide the desiredpharmaceutical effect. For instance, Compound (I) could be administeredto a warm-blooded animal orally, at a unit dose less than 1 g daily.Particularly Compound (I) could be administered to a warm-bloodedanimal, at a unit dose of less than 250 mg per day. In another aspect ofthe invention, Compound (I) could be administered to a warm-bloodedanimal, at a unit dose of less than 130 mg per day. In a further aspectof the invention, Compound (I) could be administered to a warm-bloodedanimal, at a unit dose of less than 50 mg per day.

1. A method of treating prostate cancer which comprises administering aneffective amount for treating prostate cancer ofN-(3-methoxy-5-methylpyrazin-2-yl)-2-(4-[1,3,4-oxadiazol-2-yl]phenyl)pyridine-3-sulphonamide,or a pharmaceutically acceptable salt thereof, to a warm blooded animalin need of treatment of prostate cancer.
 2. The method according toclaim 1 wherein the prostate cancer is in a metastatic state.
 3. Themethod according to claim 1 wherein the prostate cancer is in anon-metastatic state.
 4. The method according to claim 1 wherein theprostate cancer is producing bone metastases.
 5. The method according toclaim 1 wherein the warm blooded animal is man.
 6. A method for reducingabnormal proliferation in a prostate cancer cell or inducingdifferentiation of a prostate cancer cell which comprises administeringan effective amount for reducing abnormal proliferation in a prostatecancer cell or inducing differentiation of a prostate cancer cell ofN-(3-methoxy-5-methylpyrazin-2-yl)-2-(4-[1,3,4-oxadiazol-2-yl]phenyl)pyridine-3-sulphonamide,or a pharmaceutically acceptable salt thereof, to a warm blooded animalin need of reduction of abnormal proliferation of a prostate cancer cellor in need of inducing differentiation of a prostate cancer cell.
 7. Themethod according to claim 6 wherein the warm blooded animal is man.
 8. Amethod of inducing apoptosis in a prostate cancer cell which comprisesadministering an effective amount for inducing apoptosis in a prostatecancer cell ofN-(3-methoxy-5-methylpyrazin-2-yl)-2-(4-[1,3,4-oxadiazol-2-yl]phenyl)pyridine-3-sulphonamide,or a pharmaceutically acceptable salt thereof, to a warm blooded animalin need of inducing apoptosis in a prostate cancer cell.
 9. The methodaccording to claim 8 wherein the warm blooded animal is man.